Appropriate implementation of Okazaki fragment maturation during DNA replication in eukaryotic cells is a fundamental mechanism for avoidance of mutations and genome stability. During lagging strand DNA synthesis, multiple RNA primers and immediately adjoined DNA-fragments are synthesized by primase (a hetero tetramer of a RNA polymerase and DNA Pol ). However, both of the enzymes lack a proof reading function, different from the other DNA polymerases. Therefore, this initial RNA-DNA fragment (alpha- segment of the Okazaki fragment) is highly mutagenic and has to be processed by nuclease complexes. This proposal aims to define detailed molecular mechanism for the nuclease-driven RNA primer processing in eukaryotic nuclei and mitochondria. For the last funding period, we have defined the roles of several nucleases in the processes, including S. cerevisiae RNase H(35), ScRad27 or human FEN1, and exonuclease-1, and mutagenic consequences when these nucleases are defective. We have also accumulated solid evidence to demonstrate that nuclease helicase DNA2 exclusively localizes into mitochondria, and plays a vital role in RNA primer removal during mitochondrial DNA replication. These novel exciting observations prompted us to develop new and additional specific aims in this renewal application. The current proposal focuses to test a central hypothesis that 1-segment processing is a vital part of cellular mechanisms to maintain genomic integrity and prevent mutagenic stresses due to intrinsic DNA sequence obstacles and exogenous insults. Deficiency of this integrative machinery could lead to a high incidence of mutagenesis and carcinogenesis. We will further define detailed molecular mechanisms for the nuclease-driven 1-segment processing in Okazaki fragment maturation in yeast and mammalian cell systems, during replication of normal DNA sequence and repetitive DNA sequence regions, in the nucleus as well as the mitochondrion. Through a series of vigorous systematic analyses, we intend to obtain a high resolution image of how these nuclease complexes collectively work towards RNA primer processing in different scenarios and to relate in vitro and in vivo data using yeast and mammalian systems, including human cell lines and transgenic mice. Information made available from this systematic study will establish a relationship between this mechanism, unique mutagenic phenotype(s), and development of cancers.